P331: THE ONCOGENIC TRANSCRIPTION FACTOR TCF3::HLF MARKS A DISTINCT ENHANCER-PROMOTER INTERACTION NETWORK IN T(17;19) POSITIVE CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA
Authors: Valdemar Priebe, Aneta Drakul, Stefan Zeyen, Beat Bornhauser, Raffaella Santoro, Jean-Pierre Bourquin
Journal: HemaSphere (2023)

Background: TCF3::HLF is a chimeric transcription factor generated by the t(17;19) translocation harbored by a rare subtype of B-cell acute lymphoblastic leukemia (ALL). The fusion protein rewires transcriptional regulation towards a stem-like/myeloid lineage signature and promotes leukemogenesis of this resistant and fatal disease. However, the mechanisms by which TCF3::HLF regulates gene expression are not fully understood.. Aims: Based on ChIP-seq analysis revealing predominant association of the oncogenic fusion protein with enhancer and super-enhancer regions. The aim of this study was to describe the gene networks directly regulated by TCF3::HLF bound cis-regulatory sites and associated long distance genomic interactions. Ultimately the goal was to highlight gene dependencies relying on TCF3::HLF direct regulation that can be used for novel therapeutic intervention. Methods: To gain insight into the TCF3::HLF-regulated gene network, we performed TCF3::HLF targeted MNase-HiChIP in a t(17;19)-positive ALL cell line (HAL-01), in order to detect genomic interactions that are mediated by the fusion protein. HiChIP is a HiC-assay derivate that identifies long range genomic interactions captured within proximity ligated complexes bound by the protein of interest. Annotation of HiChIP identified genomic loops was performed by integrating our results with previously published differential gene expression profiles from RNA-seq following TCF3::HLF knockout and histone marker ChIP-seq in HAL-01. Selected TCF3::HLF-bound sites involved in high ranking interactions were perturbed by CRISPR and functionally investigated by competition assays. Results: We identified multiple significant interactions (n=11912, FDR <0.01), including the previously described TCF3::HLF-facilitated loop occurring between the MYC promoter and the blood enhancer cluster. We identified several genes that are differentially expressed upon TCF3::HLF knockout in HAL-01 (eg. genes significantly, p<0.005, upregulated by the fusion protein; CLSTN2, BGNT7 and POLR3G). with promoter regions in contact with TCF3::HLF-bound enhancers. The analysis of published gene expression data across ALL subtypes showed that some of these genes have higher expression in t(17;19)-positive ALLs (CLSTN2, B3GNT7), underscoring the importance of the TCF3::HLF 3D-genome interaction network in regulating genes associated with the disease. CRISPR-mediated perturbation of a TCF3::HLF-bound enhancer site with defined strong promoter-enhancer loops (MEF2C, CETN3 and POLR3G associated gene targets) caused a fitness disadvantage in HAL-01. The same perturbation had no effect in a t(1;19)-positive ALL cell line (697), which expresses the related but distinct fusion protein TCF3::PBX1. This confirms the importance TCF3::HLF hijacked enhancer sites have for the survival and growth of t(17;19)-positive ALL cells. Summary/Conclusion: To define networks of gene expression activities driven directly by the fusion protein we combined HiChIP generated 3D-genome data and omics outputs for a TCF3::HLF-positive ALL cell line. Mapping the TCF3::HLF enhancer-promoter architecture, pathway annotation and subsequent functional analysis can reveal gene dependencies of TCF3::HLF-positive ALL. These genes are primary candidates for the development of novel therapeutic approaches for this highly resistant disease entity. Keywords: B cell acute lymphoblastic leukemia, Genomics, Transcriptional regulation